Affinity chromatography is a separation technique based on specific and reversible molecular interactions between two biologically active substances. However, the development of affinity chromatography has been retarded to a great extent by the absence of suitable supports and the lack of adequate techniques for immobilizing ligands.
Affinity matrixes containing the diazonium group as the activator or coupling group are well known. The diazonium group is very reactive functionally for covalently bonding to phenols, aromatic amines and to sites on other reactive aromatic rings of ligands. The diazonium group is particularly good for selective bonding to the tyrosine group in proteins and peptides and the like and to other compounds of interest for affinity chromatography such as dyes, purine compounds and the like.
However, one of the major disadvantages to the more widespread use of diazonium matrixes in affinity chromatography applications is that such diazonium matrixes are normally quite unstable and generally must be prepared and used immediately. Additionally, because such diazonium matrixes on silica gels are normally made by an amide linkable using p-nitrobenzoyl chloride with aminopropyl silica gel, followed by reduction of the nitro groups and subsequent diazotization as disclosed in De Jong et al., J. Chem. Ed. 51(1974) 200, this produces a somewhat hydrophobic matrix that tends to bind protein non-specifically. Thus, such a diazonium matrix is somewhat unsuitable as a general affinity matrix.
Therefore, it is highly desirable that storage stable diazonium affinity matrixes be provided which are stable over extended periods and thus can be stored for future use. It is also desirable that such diazonium affinity matrixes be more hydrophilic and thus bind protein more specifically and thereby be more useful as general affinity matrixes.